Demonstration of a calcium requirement for secretory protein processing and export. Differential effects of calcium and dithiothreitol.

G Kuznetsov, M A Brostrom, C O Brostrom

Index: J. Biol. Chem. 267(6) , 3932-9, (1992)

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Abstract

HepG2 cells were employed as model system to investigate potential relationships between early protein processing and Ca2+ storage by the endoplasmic reticulum. Ca2+ was required for glycoprotein processing and export by intact cells. The processing and export of alpha 1-antitrypsin and the secretion of complement factor 3, which are glycosylated proteins, were inhibited by the Ca2+ ionophore ionomycin whereas the export of albumin, a non-glycoprotein, was little affected. Ionomycin blocked processing of alpha 1-antitrypsin at the conversion from the high mannose to the complex glycosylated form without affecting ATP or GTP contents. Pre-existing inhibition of intracellular processing of alpha 1-antitrypsin by ionomycin was fully reversible upon removal of the ionophore with fatty acid-free bovine serum albumin. This reversal required Ca2+. After reversal the arrested form of alpha 1-antitrypsin was fully converted to the mature form and exported to the medium. Inhibitions of alpha 1-antitrypsin processing and complement factor 3 secretion by the metalloendoprotease antagonist Cbz-Gly-Phe-NH2 (where Cbz is benzyloxycarbonyl) were strongest at low extracellular Ca2+ but were reduced or prevented by high extracellular Ca2+. Processing and secretion of alpha 1-antitrypsin were reduced upon incubation in low Ca2+ medium. Exposure to dithiothreitol reduced albumin export while affecting alpha 1-antitrypsin export minimally. Suppression of amino acid incorporation into total cellular proteins of HepG2 cells accompanied inhibitions of protein processing by agents depleting sequestered Ca2+ stores or by dithiothreitol. Putative control of rates of translational initiation by the endoplasmic reticulum through linkage to rates of early protein processing is discussed.